The present invention relates generally to antibodies for estrogen receptor protein and the in vitro detection and quantification of such protein through use of such antibodies. More specifically, the invention provides novel antibody preparations which have specific reactivity with estrogen recepetor and improved test methods and reagents for detection and quantification of receptor protein by means of immunological reactions.
It is generally recognized that specific estrogen-binding proteins, called "estrogen receptors" or, generically, "estrophilin" are responsible for the uptake of estrogenic hormones by certain tissues. The hormones are believed to interact with extranuclear estrophilin present in hormone dependent or "target" cells, with the "activated" estrogen-receptor complex so formed being translocated to the cell nucleus where it binds to the chromatin and in some way enhances the ability of the nucleus to synthesize certain types of RNA.
It has been determined that certain tissues, notably certain human breast cancer tissues, are estrogenic hormone "dependent" in the sense that systemic deprivation of supportive estrogen will result in regression of tissue growth and cell proliferation. As one example of this dependence, bilateral adrenalectomy can effect striking remission of advanced breast cancer in postmenopausal women and similar remissions are observed after hypophysectomy. Estrogen deprivation by surgical ablation of tissue responsible for estrogen production and/or endocrine additive therapy afford the most effective treatments presently available for advanced breast cancer. Unfortunately, less than one-half of the premenopausal patients and even a smaller fraction of postmenopausal patients respond to this type of therapy--indicating that breast cancer tissue is not always of the cellular type which is estrogenic hormone dependent. Consequently, it is significant to the prognosis and treatment of human breast cancer to be able to ascertain whether excised tumor tissue of a breast cancer patient is comprised predominantly of estrogen dependent cell types. On the basis of such information, a reasonable ablation response prediction may be made. Surgicial removal of estrogen producing glands for the purpose of estrogen deprivation may be restricted to those patients most likely to be helped by the procedure. Correlatively, other breast cancer patients can be spared the trauma of essentially useless surgery and may be placed immediately into alternative therapeutic programs such as radiation or chemotherapy.
Heretofore, the presence of estrogen dependent tissue in mammary tumor samples has principally been determined by quantitative detection of estrophilin in the sample through radiochemical assay. According to one such procedure developed by the inventors and their co-workers, radioactive (e.g., tritiated) estradiol is added to the cytosol--or supernatant fraction--of a homogenized tissue sample, and the tritiated estradiol reversibly combines with any estradiol-receptor protein present in the cytosol. The specimen is then subjected to low-salt, sucrose density gradient ultra-centrifugation and the protein-estradiol complex, being a large molecule, sediments with a characteristic velocity. A radioactive count can be used to quantify the complex. [See, e.g., Jensen, et al., J. Steroid Biochem., 7, 911-917 (1976); see also, Jensen, et al., "Estrogen Receptors and Breast Cancer Response to Adrenalectomy," National Cancer Institute Monograph, 34, 55-77 (1971)]. This procedure is carried out in the presence and in the absence of an inhibitor of the desired specific binding in order to identify and exclude any binding that is non-specific. The above analytical technique requires use of rather sophisticated, costly and uncommon ultracentrifugation apparatus, the operation of which requires a high degree of skill on the part of the laboratory worker. Other methods employed for receptor assays have similar limitations. [See, e.g., Korenman et al., J. Clin. Endocrinol. & Metab. 30, 699-645 (1970)] As a result, despite the exceptional usefulness of quantitative detection of estrophilin in prediction of response to endocrine therapy, the utilization of prior radiochemical assays is limited by scientific, geographic, and economic considerations.
It has long been recognized that immunochemical techniques for estrophilin detection would, if available, provide a simpler and less costly analytical procedure which would be susceptible to more widespread clinical use. Although suggestive evidence for the presence of antibodies to estrogen receptor in the serum of animals injected with partially purified estrophilin preparations has been reported [See, e.g., Soloff, et al., Biochem. Biophys. Res. Comm., 34, 141-147 (1969); Fox, et al., FEBS Lett., 63, 71-76 (1976); and Jensen, et al., Arch. Anat. Microscop. Morph. Exptl., 56 Suppl., 547-569 (1967)], the art has heretofore not been provided with any definitive demonstration that antibodies to estrophilin can be generated, prepared in quantity, and effectively employed in an assay for estrophilin--especially in tissue of differing species. Indeed the expectancy for success in the search for antibodies to estrophilin has been substantially diminished by past failure of allegedly highly purified progesterone receptor from oviduct tissue to give any trace of immune response in rabbits. Such findings have been supportive of wholely non-immunogenic characteristics for the proteinaceous steroid hormone receptors generally, and for estrophilin in particular.